Name: high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping
Uploaded: 2018-06-29T15:00:00
Description: Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

作者希望感谢稀有疾病模型和机制 (RDMM) 网络 (由加拿大卫生研究所 (卫生研究院) 和加拿大基因组资助)。CK 获得 UROP 奖学金 (渥太华大学) 的支持。DLJ 得到加拿大 Vanier 研究生奖学金的支持。由加拿大卫生研究院 (卫生研究院) 博士后奖学金奖资助。作者感谢美国遗传学学会批准发布这项协议, 并使用修改的补充图5的修订,等等。

有关动物的程序已获批准, 并符合加拿大动物保育委员会和渥太华大学动物保育委员会提供的动物保育指引。
1. 准备
<ol><li>通过用高压釜胶带在其内部表面上贴上9厘米的培养皿盖, 准备解剖表面。在立体声显微镜下定位。</li>
	<li>放置 3-5 天后受精 (dpf) 斑马鱼幼虫在培养皿和麻醉˜1.5 毫米 Tricaine 1x E3 胚介质 (5 毫米氯化钠, 0.17 毫米氯化钾, 0.33 毫米 CaCl2, 0.33 毫米 MgSO4, pH 7.2)?...

<ol>
	<li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), (2013).</li><li>Stewart, A. M., Kalueff, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+better+and+more+valid+animal+models+of+brain+disorders.">Developing better and more valid animal models of brain disorders.</a> Behav Brain Res. 276, 28-31 (2015).</li><li>Grone, B. P., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+epilepsy+research:+legacies+and+new+directions.">Animal models in epilepsy research: legacies and new directions.</a> Nat Neurosci. 18 (3), 339-343 (2015).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J Vis Exp. (25), e1115 (2009).</li><li>Doyon, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+targeted+gene+disruption+in+zebrafish+using+designed+zinc-finger+nucleases.">Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.</a> Nat Biotechnol. 26 (6), 702-708 (2008).</li><li>Bedell, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Genome+Editing+Using+High+Efficieny+TALENs.">In vivo Genome Editing Using High Efficieny TALENs.</a> Nature. 491 (7422), 114-118 (2012).</li><li>Sander, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+in+somatic+zebrafish+cells+using+engineered+TALENs.">Targeted gene disruption in somatic zebrafish cells using engineered TALENs.</a> Nat Biotechnol. 29 (8), 697-698 (2012).</li><li>Hwang, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+editing+in+zebrafish+using+a+CRISPR-Cas+system.">Efficient genome editing in zebrafish using a CRISPR-Cas system.</a> Nat Biotechnol. 31 (3), 227-229 (2013).</li><li>Pena, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyridoxine-Dependent+Epilepsy+in+Zebrafish+Caused+by+Aldh7a1+Deficiency.">Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.</a> Genetics. 207, 1501-1518 (2017).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nat Commun. 4, 2410 (2013).</li><li>Godoy, R., Noble, S., Yoon, K., Anisman, H., Ekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemogenetic+ablation+of+dopaminergic+neurons+leads+to+transient+locomotor+impairments+in+zebrafish+larvae.">Chemogenetic ablation of dopaminergic neurons leads to transient locomotor impairments in zebrafish larvae.</a> J Neurochem. 135, 249-260 (2015).</li><li>Scheldeman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+Disease+mTOR-related+neuropathology+in+mutant+tsc2+zebra+fish+Phenotypic,+transcriptomic+and+pharmacological+analysis.">Neurobiology of Disease mTOR-related neuropathology in mutant tsc2 zebra fish Phenotypic, transcriptomic and pharmacological analysis.</a> Neurobiol Dis. 108 (September), 225-237 (2017).</li><li>Zabinyakov, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+first+knock-out+aldh7a1+zebrafish+model+for+pyridoxine-dependent+epilepsy+using+CRISPR-Cas9+technology.">Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.</a> PLoS One. 12 (10), 1-14 (2017).</li><li>Grone, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy,+behavioral+abnormalities,+and+physiological+comorbidities+in+syntaxin-binding+protein+1+(STXBP1)+mutant+zebrafish.">Epilepsy, behavioral abnormalities, and physiological comorbidities in syntaxin-binding protein 1 (STXBP1) mutant zebrafish.</a> PLoS One. 11 (3), (2016).</li><li>Kawakami, K., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+transgenic+zebrafish.">Rapid identification of transgenic zebrafish.</a> Trends Genet. 12 (1), 9-10 (1996).</li><li>Wilkinson, R. N., Elworthy, S., Ingham, P. W., van Eeden, F. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+PCR-based+genotyping+of+larval+zebrafish+tail+biopsies.">A method for high-throughput PCR-based genotyping of larval zebrafish tail biopsies.</a> Biotechniques. 55 (6), 314-316 (2013).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+genotyping+method+for+genome-modified+animals+and+human+cells+generated+with+CRISPR/Cas9+system.">An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.</a> Sci Rep. 4, 6420 (2014).</li><li>Walsh, P. S., Metzger, D. A., Higuchi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelex+100+as+a+medium+for+simple+extraction+of+DNA+for+PCR-based+typing+from+forensic+material.">Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.</a> Biotechniques. 54 (3), 506-513 (2013).</li><li>Ewing, B., Green, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basecalling+of+automated+sequencer+traces+using+phred.+II.+Error+probabilities.">Basecalling of automated sequencer traces using phred. II. Error probabilities.</a> Genome Res. 8, 175-185 (1998).</li></ol>

基因编辑工具已经被用来精确地引入突变和转基因在过去几年斑马鱼5,6,7,8。在斑马鱼进行疾病建模时, 早期幼虫或幼年表型通常观察到9、12、13、14。这里提出的协议描述了从小斑马鱼幼虫尾活检中提取 PC...

斑马鱼 (斑马斑马)是一种脊椎动物有机体, 广泛用作疾病调查的模型, 以及治疗假说1,2,3的临床前检验。这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低费用, 并且容易地接受基因操作通过显微注射 DNA 在胚胎4。通过新的转基因和基因编辑技术的不断发展, 如锌指核酸 (ZFN)5, 核酸 (TALENs)6,7和聚集定期 Interspaced 短回文重复 (CRISPR)/CRISPR 相关 9 (Cas9) 系统8, 斑马鱼准备大大提高对几个病理条件的理解。这些技术已经被用来在斑马鱼的体细胞和生殖细胞中产生靶向性的挖空, 有效地培养转基因动物8。最近在文献中的例子包括成功地开发了癫痫的遗传模型和代谢紊乱在斑马鱼3,9,10,11,12.
随着斑马鱼基因组编辑的进展, 快速而可靠的基因分型已成为不可否认的限速步骤。在预测基因组编辑目标站点附近的特定 DNA 突变的识别是该协议的一个重要阶段。传统上, 鳍片修剪的基因分型技术通常只在青少年或成年斑马鱼身上进行。然而, 将斑马鱼饲养到成年 (> 2 月) 的时间大大延缓了研究的进展。在许多情况下, 由于关键基因的挖空 (例如疾病建模) 或功能突变导致的表型效应, 成年无法实现。此外, 一些化验需要收集幼虫, 如在 RNA 或代谢物提取, 从而涉及事先鉴定的基因型, 这可能是挑战时, 只有特殊的基因组技术可用。上述例子突出了幼虫基因分型技术的重要性, 同时也是精确的, 能够充分恢复和正常发育的幼虫。早期斑马鱼的基因分型目前似乎没有得到广泛应用;最近的一些疾病模型的出版物反映了这一点, 其中幼虫基因型是通过验尸后基因类型确定的, 而不是在实验12、13、14的执行之前分基因。本文提出了一种幼虫夹鳍策略, 旨在改进以前建立的基因分型程序。
过去, 采用蛋白酶 K 消解基因型活斑马鱼幼虫的策略导致了变聚合酶链反应 (PCR) 效率15。虽然最近公布的显微尾部活检技术提供了更有希望的结果16, 我们和其他小组无法重现的高效率和 DNA 恢复报告的作者。威尔金森等所描述的协议的一个重大倒退。16可能是尾巴组织的转移到 PCR 管。由于其显微尺寸, 很难确保鳍被正确收集和配发的吹打。为了解决这一障碍, 我们开发了一种改进的协议, 用于对大量活斑马鱼幼虫进行基因分型, 其效率为 100%9。使用 microscalpel, 鳍尾的尖端被移除并放置在一张滤纸上。该片含有组织的滤纸可以很容易地可视化, 并正确地放入 PCR 管。然后进行基因组 DNA 提取, 然后进行 PCR 扩增的感兴趣区域基因型的标本。该方法的优点有: PCR 效率高, 假阳性率低, 死亡率低。另外, 使用该协议可以对大量胚胎进行基因分型。本文所述的协议允许在 2-3 小时内用这种方法对数以百计的幼虫进行翅片修剪, 并在两天内正确识别 genotyped 鱼和尾再生。

<table>
	<tbody>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscalpel</td>
			<td>World Precision Instruments</td>
			<td>500249</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine/Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Tissue-culture plate</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate</td>
			<td>Fisher</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>S25548A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Chelex 100 sodium form</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed 1x</td>
			<td>Biotium</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>30 % Acrylamide/Bis Solution, 37.5: 1</td>
			<td>Bio-Rad&#160;</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green Master Mix, 2X</td>
			<td>Promega</td>
			<td>M7123</td>
			<td></td>
		</tr>
		<tr>
			<td>paper wipes (Kimwipes&#174; disposable wipers)</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>1kb-plus molecylar weight marker&#160;</td>
			<td>Thermo Scientific</td>
			<td>SM1343</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system (high-throughput agarose gel system)</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>vertical gel electrophoresis system Mini-PROTEAN Tetra Cell&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>1658004</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping

斑马鱼 (斑马斑马) 拥有 orthologues 已知与人类疾病相关的84% 的基因。此外, 这些动物有短的世代时间, 容易处理, 显示高繁殖率, 低成本, 并且容易地接受基因操作通过微注射 DNA 在胚胎。基因编辑工具的最新进展使斑马鱼的突变和转基因得到精确的引入。在斑马鱼的疾病建模往往导致幼虫表型和早期死亡, 这可能是挑战, 以解释, 如果基因的未知。在需要样本池的实验中也需要这种早期的基因型鉴定, 例如基因表达或质谱研究。然而, 广泛的基因型筛选受传统方法的限制, 在大多数实验室仅对成年斑马鱼或死后幼虫进行。我们解决这个问题的方法是, 采用一种分离 PCR 现成的基因组 DNA 从活斑马鱼幼虫, 可以达到早在72小时后受精 (hpf)。这一时间和成本效益的技术, 改进了从以前公布的基因分型协议, 允许鉴定的基因, 从微观鳍活检。随着幼虫的发育, 鳍迅速再生。然后, 研究人员可以利用这种高通量 PCR 的基因分型方法, 选择并提高所期望的成年期。

protocoll 的效率是通过基因分型的异型十字架 (aldh7a1+/ot100, 这里显示为aldh7a1+/) (图 3A-C)9。aldh7a1ot100突变等位基因有一个5基对 (bp) 插入的第一个编码外显子的斑马鱼aldh7a1基因导致 frameshift 和丧失功能, 由于早期停止密码子9。在多重反应中?...

Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

斑马鱼在生物医学研究中被用作可靠的遗传模型有机体, 特别是随着基因编辑技术的出现。当幼虫表型预计, DNA 提取和基因的鉴定可能是挑战。在这里, 我们描述了一个有效的基因分型程序的斑马鱼幼虫, 通过尾部修剪, 早在72小时后受精。

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

This method can help answer key questions in the biomedical field. Such as the use of zebrafish to generate genetic models of disease. The main advantage of this technique is that it allows early identification of genotypes using a simple high-throughput method.Though this method can enable extensive research using zebrafish models of genetic epilepsies, it can also be applied to other genetic disease models and any type of mutation study. Generally, individuals new to this method may have difficulties as the transection of the larval fin must be precisely performed within the pigment gap of the tail. Prior to starting the fin clipping of the zebrafish larvae, prepare the dissection surface.Tape a piece of autoclave tape across the interior surface of a nine centimeter Petri dish lid and position the lid under a stereo microscope. Prepare a P1000 pipette tip for accommodating a three dpf larva with minimal stress, by cutting off the end of the pipette tip to a diameter of two millimeters. Place three to five days post fertilization, or dpf, zebrafish larvae in a Petri dish and anesthetize in 1.5 millimolar tricaine in 1x E3 embryo medium.Using the modified P1000 micro pipette, pick up and anesthetize zebrafish larva and place it onto the positioned Autoclave tape on the Petri dish lid. Remove excess tricaine solution surrounding the larva. The area should be as dry as possible to successfully section and pick up the fin, but still wet to ensure survival.To avoid bleeding and ensure survival of the larva during fin clipping, the transection must occur within the pigment gap. Distal to the limit of the blood circulation, as indicated in this image. Working under a stereo microscope, use a micro scalpel to section the caudal fin.During the incision, apply a steady but gentle downward pressure to avoid damaging the notochord. Visualize the sectioned fin under the microscope, and position the piece on top of the tip of the micro scalpel blade. Place the micro scalpel containing the fin onto the surface of a small piece of filter paper.Due to the presence of melanocytes in the transected tissue, the fin appears as a small black spot. To ensure successful DNA extraction, it is important that a black dot marking the placement of the fin is observed on the filter paper before continuing with the protocol. Using scissors and tweezers, transfer the filter paper containing the fin to a well of a 96-well PCR plate containing 25 microliters of a 50 millimolar sodium hydroxide solution.Next, prepare a flat bottom 96-well tissue culture plate, numbering the wells according to the label of the PCR plate. Using the modified P1000 pipette, filled with 200 microliters of fresh e3 embryo medium, carefully collect the zebrafish larva using gentle pressure. Dispense the larva in the 96-well plate in the same well number as the PCR plate.Make sure the filter paper containing the fin is well submerged in the solution. To prevent cross contamination between each cut, clean the blade of the micro scalpel and the tweezers by dipping into a 70%ethanol solution. Wipe the blade with a clean paper tissue or wipe.When all larvae have been clipped, store the larvae in the 96-well plate at 28 degrees Celsius until their genotypes have been identified. Begin this procedure by sealing the 96-well PCR plate containing the fin pieces and centrifuging the samples at 1000 times G for one minute. To make sure all the filter papers are submerged in the sodium hydroxide solution.For tissue lysis, heat the samples in a thermal cycler at 95 degrees Celsius for five minutes. Followed by cooling to four degrees Celsius for 10 minutes. Next, add six microliters of 500 millimolar tris-HCL pH8.0 to each sample.Vortex the samples. Briefly centrifuge the plate at 1500 times G for five minutes at room temperature. To extract genomic DNA using this method, clip the fin as demonstrated earlier, but add the filter paper with the clipped fin to a well of a 96-well PCR plate containing 30 microliters of 5%chelating resin.After all fin pieces have been clipped, seal the 96-well PCR plate and briefly centrifuge the samples to make sure all filter papers are submerged with the chelating resin. Heat the samples in a thermal cycler at 95 degrees Celsius for 15 minutes, followed by cooling to four degrees Celsius for 10 minutes. Briefly centrifuge the samples to pellet the resin beads and obtain the DNA in the suspension.A multiplex PCR assay will enable the discrimination between homozygous mutants, heterozygotes, and wild type larvae. Set up the PCR reaction as detailed in the text protocol. Perform the PCR reactions in 96-well thermal cycler.Prepare a 1%agarose gel in sodium borate buffer, stained with 1x gel red. To facilitate the process, if possible, use a gel system that is compatible with multi channel pipettes to enable higher throughput capabilities by reducing the time to load samples. Run the gel in 1x sodium borate buffer at 250 volts for 15 minutes.Image the gel under ultraviolet light to allow discrimination of the different genotypes. Start this assay by preparing two 12%polyacrylamide gels using the 1.5 millimeter spacer plate and the 15 sample combs as described in the text protocol. Use a vertical electrophoresis system and 1x TBE buffer as the running buffer.After performing PCR as described in the text protocol, heat the PCR products at 94 degrees Celsius for five minutes, followed by cooling at four degrees Celsius for 10 minutes. Load 10 microliters per PCR sample, and eight microliters of molecular weight marker. Run the gel at 150 volts for one hour, or until the molecular weight marker almost reaches the foot line of the glass plate.Image the gel under ultraviolet light to allow discrimination of the different genotypes. This protocol was used to genotype of the offspring of a heterozygous cross, in which the mutant ileal has a 5 base pair insertion in the first coating exon of the aldh7a1 gene. The expected result from PCR amplification of both wild type and mutant ileals is a 434 BP amplicon.A 293 BP band arises from amplification of the mutant ileal, and a 195 BP band is obtained for the wild type ileal. Analysis of PCR reactions on a 1%agarose sodium borate gel identified wild type genotypes in lanes two and six, heterozygous genotypes in lanes three, four, and five, and a homozygous mutant genotype in lane one. Various mutations where genotyped by a heteroduplex melting assay.The plpbpot101 and plpbpot102 mutant ileals of the plpbp gene each let to a frame shift and early stop codon. After denaturation and annealing, the PCR fragments for heterozygous animals would contain heteroduplex and homoduplex DNA that are easily separated and visualized using polyacrylamide gel electrophoreses. Compound heterozygous mutant genotypes were identified in lanes two, four, and eight, heterozygous genotypes in lanes three, six, seven, nine, and ten, and a wild type genotype in lane five.Once mastered, fin clipping 96 larvae using this technique can be done in less than an hour, if performed properly. Following this procedure, other methods, like pooling of zebrafish larvae of the same genotype for tissue extraction, can be performed and ordered to answer additional questions, such as, biomarker investigation and disease models. After it's development, this technique paved the way for research in the field of models of metabolic epilepsy to explore disease path physiology in zebrafish.After watching this video, you should have a good understanding of how to properly genotype three day old zebrafish larvae by microdissection of a caudal fin.

Research

JoVE Journal

Genetics

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...